利用 CRISPR-Cas9 文库筛选识别膀胱癌中潜在的三级淋巴结构形成调节因子

CRISPR-Cas9 activation screening identifies candidate chemokine regulators of ter- tiary lymphoid structure formation in bladder cancer

摘要

目的：通过 CRISPR-Cas9 文库篮选影响三级淋巴结构（tertiary lymphoid structure，TLS）形成的调节因子，鉴定潜在的关键调控分子，为提升膀胱癌免疫治疗疗效提供新靶点。方法：基于小鼠全基因组文库，筛选44个趋化因子相关基因，设计并构建靶向这些基因的单向导 RNA（single-guide RNA，sgRNA）文库，每个基因配置3条sgRNA。利用慢病毒包装系统将文库质粒感染 HEK293T 细胞，获得慢病毒文库并感染小鼠膀胱癌细胞系 MB49。通过嘌呤霉素筛选获得稳定过表达趋化因子的 MB49-mCherry 细胞株。将细胞接种于 C57BL/6小鼠腹腔，建立膀胱癌移植瘤模型，观察肿瘤生长情况。 3 周后剥离肿瘤组织，提取基因组 DNA 进行高通量测序，分析 sgRNA 富集情况，筛选差异表达的趋化因子基因。同时，通过免疫组织化学染色检测 TLS 标志物 CD20 和 CD3，评估 TLS 数量、分布及成熟度。对筛选出的候选基因进行单独体内验证，进一步确认其对 TLS 形成的影响。结果：成功构建了包含132条 sgRNA 的细胞因子基因文库，覆盖44个趋化因子基因。慢病毒感染后获得稳定表达文库的 MB49-mCherry细胞株，流式分选获得失活的 Cas9 阳性单克隆细胞株，确保后续实验的均一性和可重复性。小鼠腹腔肿瘤实验显示，实验组肿瘤组织中 TLS 数量较对照组明显增多，主要分布于肿瘤边缘。高通量测序结果显示，与对照组相比，实验组中 Cxcl16 sgDNA 显著富集，Ccl20 和 Cx3cl1 sgDNA 则较初始有所下降（ ^1,2*）。进一步通过腹腔注射分别验证各趋化因子的作用，发现给予 CX3CL1 组肿瘤中 TLS 数量减少，提示 CX3CL1 可能负向调控 TLS 形成。免疫组织化学结果显示，CX3CL1 处理组肿瘤组织中 CD20 阳性 B 细胞和 CD3 阳性 T 细胞聚集减少，TLS 结构不完整。结论：通过 CRISPR-Cas9 文库筛选结合体内验证，初步鉴定出 CX3CL1 可能为调控膀胱癌 TLS 形成的负向调节因子。CX3CL1 高表达与 TLS 数量减少相关，提示其在膀胱癌免疫微环境中可能发挥抑制作用，这为深人理解膀胱癌中 TLS 形成的分子机制提供了新的线索和研究方向，但 CX3CL1 是否可作为免疫治疗靶点，仍需后续结合临床样本、多维度分子机制解析及免疫治疗响应相关性研究进行进一步验证。

Abstract

Objective：To identify the cytokine genes influencing the formation of tertiary lymphoid structures（TLS）through CRISPR-Cas9 library screening，and to discover potential key regulatory mole- cules，providing new targets for enhancing the efficacy of bladder cancer immunotherapy. Methods： Based on a mouse whole-genome library， 44 chemokine-related genes were identified，and an single- guide RNA（sgRNA）library targeting these genes was designed and constructed，with three sgRNAs assigned to each gene.Using a lentiviral packaging system，the library plasmids were used to transfect HEK293T cells to generate a lentiviral library，which was then used to infect the mouse bladder cancer cell line MB49.Purinomycin selection was performed to obtain the MB49-mCherry cell line stably over- expressing chemokines.The cells were inoculated into the peritoneal cavity of C57BL/6 mice to establish a bladder cancer xenograft model，and tumor growth was monitored.Three weeks later，tumor tissue was excised，genomic DNA was extracted for high-throughput sequencing，and sgRNA enrichment was ana- lyzed to screen for differentially expressed cytokine genes.Concurrently，immunohistochemical staining was performed to detect TLS markers CD20 and CD3，and the number，distribution，and maturity of TLS were assessed.The selected candidate genes were validated individually in vivo to further confirm their impact on TLS formation. Results：We successfully constructed a cytokine gene library containing 132 sgRNAs，covering 44 chemokine genes.Following lentiviral infection，we obtained the MB49-mCherry cell line，which stably expressed the library，and isolated dead Cas9-positive monoclonal cell lines via flow cytometry to ensure the homogeneity and reproducibility of subsequent experiments.Intratumoral tumor experiments in mice revealed that the number of TLS cells in the experimental group was signifi- cantly higher than in the control group，primarily distributed at the tumor margins.High-throughput sequencing results showed that，compared with the control group，in the experimental group，Cxcl16 sgDNA was significantly enriched，while Ccl20 and Cx3lc1 sgDNA levels decreased compared with baseline （）.Further validation of the individual roles of each factor via intraperitoneal injection revealed that the number of TLSs in tumors decreased in the group treated with the CX3 CL1 chemokine，sugges- ting that CX3 CL1 might negatively regulate TLS formation.Immunohistochemical results showed that in the CX3CL1-treated group，the aggregation of CD20-positive B cells and CD3-positive T cells in the tumor tissue was reduced，and the TLS structure was incomplete. Conclusion：Through CRISPR-Cas9 library screening combined with in vivo validation，this study successfully identified CX3CL1 as a poten- tial negative regulator of TLS formation in bladder cancer.High CX3CL1 expression was associated with a reduction in TLS numbers，suggesting that it might exert an inhibitory role in the immune microenviron- ment of bladder cancer.This finding provides new clues and research directions for understanding the mo- lecular mechanisms of TLS formation in bladder cancer.However，whether CX3CL1 can serve as an im- munotherapeutic target remains to be further validated through clinical specimen analysis，multidimen- sional mechanistic investigation，and immunotherapy response correlation studies.

关键词

膀胱肿瘤 / CRISPR-Cas 系统 / 三级淋巴结构 / 基因文库 / 炎症趋化因子

Key words

Urinary bladder neoplasms / CRISPR-Cas systems / Tertiary lymphoid structures / Gene li-brary / Chemokines

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基金资助

科技创新2030—＂癌症、心脑血管、呼吸和代谢性疾病防治研究＂国家科技重大专项(2024ZD0525700)

国家自然科学基金(82471866)

国家自然科学基金(82472912)

北京市自然科学基金(L252190)